Liposomes or lipid vesicles are onion-like structures comprising a series of bimolecular lipid layers spaced from one another by an aqueous solution, the outermost layer being lipid. Liposomes have been advantageously used to encapsulate biologically active materials for a variety of uses. The prior art describes a number of techniques for producing synthetic liposomes. Most of these techniques relate to the formation of unilamellar vesicles. For example, U.S. Pat. No. 4,078,052 - Papahadjopoulos describes a procedure for producing large unilamellar vesicles (LUV). This particular procedure, however, is restricted to the lipid phosphalidyserine which was found to uniquely form the intermediate cochleate structure, apparently essential to the formation of the large lipid vesicles, in the presence of calcium cations.
A variety of other techniques have also been disclosed for producing small unilamellar vesicles (SUV). In one approach, a mixture of the lipid and an aqueous solution of the material to be encapsulated is warmed and then subjected to vigorous agitation and ultrasonic vibration. In another approach, U.S. Pat. No. 4,089,801 - Schneider, a mixture of a lipid, an aqueous solution of the material to be encapsulated, and a liquid which is insoluble in water is subjected to ultrasonication, whereby aqueous globules encased in a monomolecular lipid layer are formed dispersed in the water-insoluble liquid. The lipid vesicles are then formed by combining the first dispersion with a second aqueous fluid and then subjecting the mixture to centrifugation, whereby the globules are forced through the monomolecular lipid layer dividing the two phases, thereby forming the bimolecular lipid layer characteristic of liposomes. In still another approach, (O. Zumbuehl and H. G. Weder, Biochim. Biophys. Acta., 640: 252-262, 1981), the lipids and additives are solubilized with detergents by agitation or sonication, yielding defined mixed micelles. The detergents are then removed by dialysis.
Two alternate methods for the preparation of small unilamellar vesicles (SUV) that avoid the need for sonication are the ethanol injection technique (S. Batzri and E. D. Korn, Biochim. Biophys. Acta 198: 1015-1019, 1973) and the ether-infusion technique (D. Deamer and A. D. Bangham, Biochim. Biophys. Acta 443: 629-634, 1976). In these processes, the organic solution of lipids is rapidly injected into a buffer solution where it spontaneously forms liposomes.
A more recent method for preparing large unilamellar lipid vesicles (LUV) is the reverse phase evaporation technique described in U.S. Pat. No. 4,235,871 - Papahadjopoulos. This technique consists of forming a water-in-oil emulsion of (a) the lipids in an organic solvent and (b) the substances to be encapsulated in an aqueous buffer solution. Removal of the organic solvent under reduced pressure produces a mixture having a gel-like character which can then be converted to the lipid vesicles by agitation or by dispersion in an aqueous media.
U.S. Pat. No. 4,016,100 - Suzuki et al describes still another method of entrapping certain biologically active materials in unilamellar lipid vesicles by freezing an aqueous phospholipid dispersion of the biologically active materials and lipids.
For a comprehensive review of methods for preparing liposomes refer to a recent publication by Szoka and Papahadjopoulos (Ann. Rev. Biophys. Bioeng. 9: 467-508, 1980).
Methods for producing multilamellar lipid vesicles (MLV), are described by Bangham et al (J. Mol. Biol. 13: 238-252, 1965) and by Mezei and Gulasekharam, (Life Sci., 26: 1473-1477, 1980). The lipids and lipophilic substances are first dissolved in an organic solvent. The solvent is then removed under reduced pressure by rotary evaporation. The lipid residue forms a thin film on the wall of the container. Upon the addition of an aqueous solution, generally containing electrolytes or hydrophilic biologically active materials, large multilamellar lipsomes are formed. Small unilamellar vesicles can be prepared by sonication of the large multilamellar vesicles.
Most of these processes suffer from either low encapsulation efficiency or limitations in the types of materials that can be encapsulated or both. For example, most of these processes are limited to the encapsulation of hydrophilic materials, and cannot efficiently accommodate the encapsulation of lipophilic substances. Moreover, all of the currently available procedures, except the ones described by Bangham et al and by Mezei and Gulasekharam, are only suitable for the encapsulation of biologically active materials in oligolamellar, or unilamellar liposomes.
It is an object of the present invention to provide a process for encapsulating biologically active materials in large multilamellar lipid vesicles.
It is another object of this invention to provide a method for encapsulating biologically active materials that results in a significant increase in the encapsulation efficiency thereof.
It is still another object of this invention to provide a method of encapsulating biologically active materials in large multilamellar lipid vesicles that is not limited with respect to the material to be encapsulated and can efficiently accommodate both lipophilic and hydrophilic substances.
It is a further object of this invention to provide a procedure for encapsulating biologically active materials in a multilamellar lipid vesicle that can be conducted on a larger scale relative to prior art procedures.